N-(5-isoxazolyl)biphenylsulfonamides, N-(3-isoxazolyl)biphenylsulfonamides and derivatives thereof that modulate the activity of endothelin

ABSTRACT

N-(5-isoxazolyl)biphenylsulfonamides and N-(3-isoxazolyl)biphenylsulfonamides and methods for modulating or altering the activity of the endothelin family of peptides are provided. In particular, N-(5-isoxazolyl)biphenylsulfonamides and N-(3-isoxazolyl)biphenylsulfonamides and methods for inhibiting the binding of an endothelin peptide to an endothelin receptor or increasing the activity of endothelin peptides by contacting the receptor with a sulfonamide are provided. Methods for treating endothelin-mediated disorders by administering effective amounts of one or more of these sulfonamides or prodrugs thereof that inhibit or increase the activity of endothelin are also provided.

RELATED APPLICATIONS

This application is a continuation-in-part of the following applications: U.S. application Ser. No. 08/100,565 to Chan et al., filed Jul. 30, 1993, now abandoned, entitled "N-(5-ISOXAZOLYL)-SULFONAMIDES AND DERIVATIVES THEREOF THAT MODULATE THE ACTIVITY OF ENDOTHELIN"; U.S. application Ser. No. 08/100,125 to Chan et al., filed July 30, 1993, now abandoned, entitled "N-(3-ISOXAZOLYL)-SULFONAMIDES AND DERIVATIVES THEREOF THAT MODULATE THE ACTIVITY OF ENDOTHELIN", and U.S. application Ser. No. 08/065,202, to Chan, filed May 20, 1993, now abandoned, entitled "SULFONAMIDES AND DERIVATIVES THEREOF THAT MODULATE THE ACTIVITY OF ENDOTHELIN".

U.S. application Ser. Nos. 08/100,565 and 08/100,125 are continuation-in-part applications of U.S. application Ser. No. 08/065,202.

The subject matter of U.S. application Ser. Nos. 08/100,565, 08/100,125, 08/065,202 are each incorporated herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to the compounds that modulate the activity of the endothelin family of peptides. In particular, the invention relates to the use of sulfonamides and sulfonamide pro-drugs as endothelin agonists and antagonists.

BACKGROUND OF THE INVENTION

The vascular endothelium releases a variety of vasoactive substances, including the endothelium-derived vasoconstrictor peptide, endothelin (ET) (see, e.g., Vanhoutte et al. (1986) Annual Rev. Physiol. 48: 307-320; Furchgott and Zawadski (1980) Nature 288: 373-376). Endothelin, which was originally identified in the culture supernatant of porcine aortic endothelial cells (see, Yanagisawa et al. (1988) Nature 332: 411-415), is a potent twenty-one amino acid peptide vasoconstrictor. It is the most potent vasopressor known and is produced by numerous cell types, including the cells of the endothelium, trachea, kidney and brain. Endothelin is synthesized as a two hundred and three amino acid precursor preproendothelin that contains a signal sequence which is cleaved by an endogenous protease to produce a thirty-eight (human) or thirty-nine (porcine) amino acid peptide. This intermediate, referred to as big endothelin, is processed in vivo to the mature biologically active form by a putative endothelin-converting enzyme (ECE) that appears to be a metal-dependent neutral protease (see, e.g., Kashiwabara et al. (1989) FEBS Lttrs. 247: 337-340). Cleavage is required for induction of physiological responses (see, e.g., von Geldern et al. (1991) Peptide Res. 4: 32-35). In porcine aortic endothelial cells, the thirty-nine amino acid intermediate, big endothelin, is hydrolyzed at the Trp²¹ -Val²² bond to generate endothelin-1 and a C-terminal fragment. A similar cleavage occurs in human cells from a thirty-eight amino acid intermediate. Three distinct endothelin isopeptides, endothelin-1, endothelin-2 and endothelin-3, that exhibit potent vasoconstrictor activity have been identified.

The family of three isopeptides endothelin-1, endothelin-2 and endothelin-3 are encoded by a family of three genes (see, Inoue et al. (1989) Proc. Natl. Acad. Sci. USA 86: 2863-2867; see, also Saida et al. (1989) J. Biol. Chem. 264: 14613-14616). The nucleotide sequences of the three human genes are highly conserved within the region encoding the mature 21 amino acid peptides and the C-terminal portions of the peptides are identical. Endothelin-2 is (Trp⁶,Leu⁷) endothelin-1 and endothelin-3 is (Thr²,Phe⁴,Thr⁵,Tyr⁶,Lys⁷,Tyr¹⁴) endothelin-1. These peptides are, thus, highly conserved at the C-terminal ends.

Release of endothelins from cultured endothelial cells is modulated by a variety of chemical and physical stimuli and appears to be regulated at the level of transcription and/or translation. Expression of the gene encoding endothelin-1 is increased by chemical stimuli, including adrenaline, thrombin and Ca²⁺ ionophore. The production and release of endothelin from the endothelium is stimulated by angiotensin II, vasopressin, endotoxin, cyclosporine and other factors (see, Brooks et al. (1991) Eur. J. Pharm. 194:115-117), and is inhibited by nitric oxide. Endothelial cells appear to secrete short-lived endothelium-derived relaxing factors (EDRF), including nitric oxide or a related substance (Palmer et al. (1987) Nature 327: 524-526), when stimulated by vasoactive agents, such as acetylcholine and bradykinin. Endothelin-induced vasoconstriction is also attenuated by atrial natriuretic peptide (ANP).

The endothelin peptides exhibit numerous biological activities in vitro and in vivo. Endothelin provokes a strong and sustained vasoconstriction in vivo in rats and in isolated vascular smooth muscle preparations; it also provokes the release of eicosanoids and endothelium-derived relaxing factor (EDRF) from perfused vascular beds. Intravenous administration of endothelin-1 and in vitro addition to vascular and other smooth muscle tissues produce long-lasting pressor effects and contraction, respectively (see, e.g., Bolger et al. (1991) Can. J. Physiol. Pharmacol. 69: 406-413). In isolated vascular strips, for example, endothelin-1 is a potent (EC₅₀ =4×10⁻¹⁰ M), slow acting, but persistent, contractile agent. In vivo, a single dose elevates blood pressure in about twenty to thirty minutes. Endothelin-induced vasoconstriction is not affected by antagonists to known neurotransmitters or hormonal factors, but is abolished by calcium channel antagonists. The effect of calcium channel antagonists, however, is most likely the result of inhibition of calcium influx, since calcium influx appears to be required for the longlasting contractile response to endothelin.

Endothelin also mediates renin release, stimulates ANP release and induces a positive inotropic action in guinea pig atria. In the lung, endothelin-1 acts as a potent bronchoconstrictor (Maggi et al. (1989) Eur. J. Pharmacol. 160: 179-182). Endothelin increases renal vascular resistance, decreases renal blood flow, and decreases glomerular filtrate rate. It is a potent mitogen for glomerular mesangial cells and invokes the phosphoinoside cascade in such cells (Simonson et al. (1990) J. Clin. Invest. 85: 790-797).

There are specific high affinity binding sites (dissociation constants in the range of 2-6×10⁻¹⁰ M) for the endothelins in the vascular system and in other tissues, including the intestine, heart, lungs, kidneys, spleen, adrenal glands and brain. Binding is not inhibited by catecholamines, vasoactive peptides, neurotoxins or calcium channel antagonists. Endothelin binds and interacts with receptor sites that are distinct from other autonomic receptors and voltage dependent calcium channels. Competitive binding studies indicate that there are multiple classes of receptors with different affinities for the endothelin isopeptides. The sarafotoxins, a group of peptide toxins from the venom of the snake Atractaspis eingadensis that cause severe coronary vasospasm in snake bite victims, have structural and functional homology to endothelin-1 and bind competitively to the same cardiac membrane receptors (Kloog et al. (1989) Trends Pharmacol, Sci. 10: 212-214).

Two distinct endothelin receptors, designated ET_(A) and ET_(B), have been identified and DNA clones encoding each receptor have been isolated (Arai et al. (1990) Nature 348: 730-732; Sakurai et al. (1990) Nature 348: 732-735). Based on the amino acid sequences of the proteins encoded by the cloned DNA, it appears that each receptor contains seven membrane spanning domains and exhibits structural similarity to G-protein-coupled membrane proteins. Messenger RNA encoding both receptors has been detected in a variety of tissues, including heart, lung, kidney and brain. The distribution of receptor subtypes is tissue specific (Martin et al. (1989) Biochem. Biophys. Res. Commun. 162: 130-137). ET_(A) receptors appear to be selective for endothelin-1 and are predominant in cardiovascular tissues. ET_(B) receptors are predominant in noncardiovascular tissues, including the central nervous system and kidney, and interact with the three endothelin isopeptides (Sakurai et al. (1990) Nature 348: 732-734). In addition, ET_(A) receptors occur on vascular smooth muscle, are linked to vasoconstriction and have been associated with cardiovascular, renal and central nervous system diseases; whereas ET_(B) receptors are located on the vascular endothelium, linked to vasodilation (Takayanagi et al. (1991) FEBS Lttrs. 282: 103-106) and have been associated with bronchoconstrictive disorders.

By virtue of the distribution of receptor types and the differential affinity of each isopeptide for each receptor type, the activity of the endothelin isopeptides varies in different tissues. For example, endothelin-1 inhibits ¹²⁵ I-labelled endothelin-1 binding in cardiovascular tissues forty to seven hundred times more potently than endothelin-3. ¹²⁵ I-labelled endothelin-1 binding in non-cardiovascular tissues, such as kidney, adrenal gland, and cerebellum, is inhibited to the same extent by endothelin-1 and endothelin-3, which indicates that ET_(A) receptors predominate in cardiovascular tissues and ET_(B) receptors predominate in non-cardiovascular tissues.

Endothelin plasma levels are elevated in certain disease states. Endothelin-1 plasma levels in healthy individuals, as measured by radioimmunoassay (RIA), are about 0.26-5 pg/ml. Blood levels of endothelin-1 and its precursor, big endothelin, are elevated in shock, myocardial infarction, vasospastic angina, kidney failure and a variety of connective tissue disorders. In patients undergoing hemodialysis or kidney transplantation or suffering from cardiogenic shock, myocardial infarction or pulmonary hypertension levels are as high as 35 pg/ml have been observed (see, Stewart et al. (1991) Annals Internal Med. 114: 464-469). Because endothelin is likely to be a local, rather than a systemic, regulating factor, it is probable that the levels of endothelin at the endothelium/smooth muscle interface are much higher than circulating levels.

Endothelin agonists and antagonists

Because endothelin is associated with certain disease states and is implicated in numerous physiological effects, compounds that can interfere with or potentiate endothelin-associated activities, such as endothelin-receptor interaction and vasoconstrictor activity, are of interest. A limited number of compounds that exhibit endothelin antagonistic activity have been identified. In particular, a fermentation product of Streptomyces misakiensis, designated BE-18257B, has been identified as an ET_(A) receptor antagonist. BE-18257B is a cyclic pentapeptide, cyclo(D-Glu-L-Ala-allo-D-Ile-L-Leu-D-Trp), which inhibits ¹²⁵ I-labelled endothelin-1 binding in cardiovascular tissues in a concentration-dependent manner (IC₅₀ 1.4 μM in aortic smooth muscle, 0.8 μM in ventricle membranes and 0.5 μM in cultured aortic smooth muscle cells), but fails to inhibit binding to receptors in tissues in which ET_(B) receptors predominate at concentrations up to 100 μM. Cyclic pentapeptides related to BE-18257B, such as cyclo(D-Asp-Pro-D-Val-Leu-D-Trp)(BQ-123), have been synthesized and shown to exhibit activity as ET_(A) receptor antagonists (see, U.S. Pat. No. 5,114,918 to Ishikawa et al.; see, also, EP A1 0 436 189 to BANYU PHARMACEUTICAL CO., LTD (Oct. 7, 1991)). Studies that measure the inhibition by these cyclic peptides of endothelin-1 binding to endothelin-specific receptors indicate that these cyclic peptides bind preferentially to ET_(A) receptors.

The analog [Ala¹,3,11,15 ]endothelin-1, in which the four Cys residues are replaced with Ala, inhibits ¹²⁵ I-endothelin-1 binding to cerebral membranes, in which ET_(B) receptors predominate (Hiley et al. (1989) Trends Pharmacol. Sci 10: 47-49). This peptide and certain truncated forms of endothelin-1 elicit endothelium-dependent vasorelaxation of precontracted porcine pulmonary arteries to an extent that parallels the respective binding affinities of each form for ET_(B) (Saeki et al. (1991) Biochem. and Biophys Res. Commun. 179: 286-292).

Endothelin Antagonists and Agonists as Therapeutic Agents

In view of the numerous physiological effects of endothelin and its apparent association with certain diseases, endothelin is believed to play a critical role in pathophysiological conditions, including hypertension, atherosclerosis, other vascular disorders, gastrointestinal disorders, renal failure, asthma, pulmonary hypertension, endotoxin shock, coronary vasospasm, cerebral vasospasm and others (see, e.g., Saito et al. (1990) Hypertension 15: 734-738; Tomita et al. (1989) N.Engl.J. Med. 321: 1127; Doherty (1992) J. Med. Chem. 35: 1493-1508; Morel et al. (1989) Eur. J. Pharmacol. 167: 427-428). Because endothelin is associated with these and other disease states, more detailed knowledge of the function and structure of the endothelin peptide family should provide insight in the progression and treatment of such conditions.

To aid in gaining this understanding, there is a need to identify compounds that modulate or alter endothelin activity. Compounds that modulate endothelin activity, particularly compounds that act as specific antagonists or agonists, may not only aid in elucidating the function of endothelin, but may be therapeutically useful. In particular, compounds that specifically interfere with the interaction of endothelin peptides with the ET_(A), ET_(B) or other receptors should may aid in the design of therapeutic agents, and may be useful as disease specific therapeutic agents.

Therefore, it is an object herein to provide compounds that have the ability to modulate the biological activity of one or more of the endothelin isopeptides. It is another object to provide compounds that have use as specific endothelin antagonists, It is also an object to use compounds that specifically interact with or inhibit the interaction of endothelin peptides with ET_(A) or ET_(B) receptors as therapeutic agents for the treatment of endothelin-mediated diseases and disorders.

SUMMARY OF THE INVENTION

Compounds and methods for inhibiting the binding of an endothelin peptide to ET_(A) or ET_(B) receptors and compounds and methods for increasing endothelin receptor-mediated activity of an endothelin peptide are provided. The methods are effected by contacting the receptors with one or more sulfonamides prior to, simultaneously with, or subsequent to contacting the receptors with an endothelin peptide. The sulfonamides are substituted or unsubstituted monocyclic or polycyclic aromatic or heteroaromatic sulfonamides, such as benzene sulfonamides and naphthalene sulfonamides.

The sulfonamides have formula I: ##STR1## in which Ar¹ is a substituted or unsubstituted aryl group with one or more substituents, including an alkyl group, an aryl group, a substituted aryl group, a nitro group, an amino group or a halide. Ar¹ is preferably a five or six membered substituted or unsubstituted aromatic or heteroaromatic ring, including, 3- or 5- isoxazolyl, 2-thiazolyl, 2-pyrimidinyl, or substituted benzene groups, including aryloxy substituted benzene groups.

Ar¹ is more preferably selected from:

and R is selected from H, NH₂, alkyl, halide, pseudohalide, alkylcarbonyl, formyl, an aromatic or heteroaromatic group, alkoxyalkyl, alkylamino, alkylthio, arylcarbonyl, aryloxy, arylamino, arylthio, haloalkyl, haloaryl, carbonyl, in which the aryl and alkyl portions are straight or branched chains of from about 1 up to about 10-12 carbons, preferably, 1 to about 5 or 6 carbons and the alkyl or aryl portions may be unsubstituted or further substituted with other groups, particularly alkyl groups, such as dialkylaminoalkyl groups. R is preferably H, NH₂, halide, CH₃, CH₃ O or another aromatic group. Ar² is alkyl, alkenyl, or is a group selected from: ##STR2## in which R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ are each selected independently from among (i), (ii), (iii) or (iv) as follows:

(i) R³, R⁴, R⁵, R⁶, and R⁷ are each selected independently from among H, NHOH, NH₂, NO₂, N₃, aminoalkyl, alkylamino, dialkylamino, carboxyl, carbonyl, hydroxyl, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, alkylamino, alkylthio, alkyalkoxy, alkylsulfinyl, alkylsulfonyl, aryloxy, arylalkoxy, aryloxy, arylamino, arylthio, arylsulfinyl, arylsulfonyl, haloalkyl, haloaryl, haloalkoxy, alkoxycarbonyl, alkylcarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amido, substituted or unsubstituted ureido, where the alkyl, alkenyl, alkynyl portions are straight or branched chains, acyclic or cyclic, of from about 1 up to about 10 carbons, preferably, 1 to about 5 or 6 carbons and the aryl portions contain from 3 up to about 10 carbons, preferably 3 to 6 carbons; R⁸, R⁹ and R¹⁰ are each independently selected from H, NH₂, NO₂, alkyl and halide; X is O, S, NH or NR¹¹ in which R¹¹ is selected from H, alkyl, alkylcarbonyl or formyl; and n is from 0 up to about 6, and preferably 0 up to about 3 and more preferably 0, 1 or 2; or, alternatively,

(ii) R⁴ and R⁷ together are substituted or unsubstituted 1,3-butadienyl, 4-dimethylamino-1,3 butadiene, 1-chloro-1,3-butadiene, 4-dimethylamino-1,3-butadienyl, 1-aza-1,3-butadienyl or 2-aza-1,3-butadienyl groups; and n, X, R³, R⁵, R⁶, R⁸, R⁹, R¹⁰ and R¹¹ are as defined in (i) above; or alternatively,

(iii) R⁷ and R³ together are substituted or unsubstituted 1,3-butadienyl, 4-dimethylamino-1,3 butadiene, 1-chloro-1,3-butadiene, 4-dimethylamino-1,3-butadienyl, 1-aza-1,3-butadienyl or 2-aza-1,3-butadienyl groups; and n, X, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰ and R¹¹ are as defined in (i) above; or alternatively,

(iv) R³, R⁵, and R⁷ are H; R⁸, R⁹, R¹⁰ and R¹¹ are as defined in (i); and R⁴ and R⁶ are each independently selected from alkyl, alkoxy, halide amino alkyl, alkylaminoalkyl or dialkylaminoalkyl, which are unsubstituted or substituted with groups, such as any set forth for R⁸, R⁹, R¹⁰ and R¹¹, and in which the alkyl and alkoxy groups contain from 1 to 10, preferably 1 to 6 carbons, and are straight or branched chains.

Thus, in embodiments herein, Ar² is a substituted or unsubstituted group selected from among the following: naphthyl, phenyl, biphenyl, quinolyl, styryl, thiophenyl, furanyl, isoquinolyl, pyrrolyl, benzofuranyl, thionaphthalyl, indolyl, alkyl, and alkenyl.

The preferred compounds have formula II: ##STR3## in which R¹ and R² are either (i), (ii) or (iii) as follows: (i) R¹ and R² are each independently selected from H, NH₂, NO₂, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkyloxy, haloalkyl, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylsufinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amido, substituted or unsubstituted ureido, in which the alkyl, alkenyl and alkynyl portions contain from 1 up to about 14 carbon atoms and are either straight or branched chains or cyclic, and the aryl portions contain from about 4 to about 16 carbons; or,

(ii) R¹ and R² together form --(CH₂)_(n), where n is 1 to 6, preferably 1 to 4; or,

(iii) R¹ and R² together form --CH═CH--;

Ar² is as described above in which R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ are each selected independently from among (i), (ii), (iii) or (iv) as follows:

(i) R³, R⁴, R⁵, R⁶, and R⁷ are each selected independently from among H, NHOH, NH₂, NO₂, N₃, aminoalkyl, alkylamino, dialkylamino, carboxyl, carbonyl, hydroxyl, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, alkylamino, alkylthio, alkoxyalkyl, alkylsulfinyl, alkylsulfonyl, aryloxy, arylalkoxy, aryloxy, arylamino, arylthio, arylsulfinyl, arylsulfonyl, haloalkyl, haloaryl, haloalkoxy, alkoxycarbonyl, alkylcarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amido, substituted or unsubstituted ureido, where the alkyl, alkenyl, alkynyl portions are straight or branched chains, acyclic or cyclic, of from about 1 up to about 10 carbons, preferably, 1 to about 5 or 6 carbons and the aryl portions contain from 3 up to about 10 carbons, preferably 3 to 6 carbons; R⁸, R⁹ and R¹⁰ are each independently selected from H, NH₂, NO₂, alkyl and halide; X is O, S, NH or NR¹¹ in which R¹¹ is selected from H, alkyl, alkylcarbonyl or formyl; and n is from 0 up to about 6, and preferably 0 up to about 3 and more preferably 0, 1 or 2; or, alternatively,

(ii) R⁴ and R⁷ together are substituted or unsubstituted 1,3-butadienyl, 4-diamino-1,3-butadienyl, 4-dimethylamino-1,3 butadiene, 1-chloro-1,3-butadiene, 1-aza-1,3-butadienyl or 2-aza-1,3-butadienyl groups; and n, X, R³, R⁵, R⁶, R⁸, R⁹, R¹⁰ and R¹¹ are as defined in (i) above; or alternatively,

(iii) R⁷ and R³ together are substituted or unsubstituted 1, 3-butadienyl, 4-diamino-1,3-butadienyl, 4-dimethylamino-1,3 butadiene, 1-chloro-1,3-butadiene, 1-aza-1,3-butadienyl or 2-aza-1,3-butadienyl groups; and n, X, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰ and R¹¹ are as defined in (i) above, or alternatively,

(iv) R³, R⁵, and R⁷ are H; R⁸, R⁹, R¹⁰ and R¹¹ are as defined in (i); and R⁴ and R⁶ are each independently selected from alkyl, alkoxy, halide, aminoalkyl, dialkylamino and dialkylaminoalkyl, which are unsubstituted or substituted with groups, such as those set forth for any of R⁸, R⁹, R¹⁰ and R¹¹, and in which the alkyl and alkoxy groups contain from 1 to 10, preferably 1 to 6 carbons, and are straight or branched chains.

In preferred embodiments, R¹ and R² are selected independently from among lower alkyl, lower alkenyl, lower alkynyl, lower haloalkyl, halide, pseudohalide or H; Ar² is as defined above, X is NH, S or O, n is 0 or 1, and either:

(i) R⁴, R⁵, R⁶ and R⁷ are each independently selected from H, lower alkyl, NH₂, NO₂, halide, pseudohalide; and R³ is selected from H, NHOH, NH₂, NO₂, N₃, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, alkylamino, alkylthio, alkyalkoxy, alkylsulfinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylsulfinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amido, substituted or unsubstituted ureido, where the alkyl, alkenyl, alkynyl portions are straight or branched chains of from about 1 up to about 5 or 6 carbons and the aryl portions contain from 1 up to about 6 carbons; R¹⁰ is H; R¹¹ is H or CH₃ ; R⁸ and R⁹ are each selected independently from among H, NO₂, NH₂ and halide; or

(ii) R⁴ and R⁷ together form 1, 3-butadienyl, 4-diamino-1,3-butadienyl, 4-chloro-1,3-butadienyl, or 1-aza-1,3-butadienyl; and R³, R⁵, R⁶, R⁸, R⁹, R¹⁰ and R¹¹ are defined as in (i) of this embodiment; or

(iii) R⁷ and R³ together form 1,3-butadienyl, 4-diamino-1,3-butadienyl, 3-chloro-1,3-butadienyl or 1-aza-1,3-butadienyl; and R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰ and R¹¹ are as defined in (i) of this embodiment; or

(iv) R³, R⁵, and R⁷ are H; R⁸, R⁹, R¹⁰ and R¹¹ are as defined in (i); and R⁴ and R⁶ are each independently selected from alkyl, alkoxy, halide, aminoalkyl, dialkylamino and dialkylaminoalkyl, which are unsubstituted or substituted with groups, such as those set forth for any of R⁸, R⁹, R¹⁰ and R¹¹, and in which the alkyl and alkoxy groups contain from 1 to 6 carbons, and are straight or branched chains.

In the embodiments herein, Ar¹ is N-(5-isoxazolyl) or N-(3-isoxazolyl) and Ar² is a substituted benzene group in which R³, R⁴, R⁵, R⁶ and R⁷ are selected independently from phenyl, substituted phenyl or H. Thus, Ar² is unsubstituted or substituted biphenyl group of formula: ##STR4## in which: (i) R¹² and R¹³ are independently selected from H, OH NH S, NH₂, NO₂, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, dialkylamino, alkylthio, alkyloxy, haloalkyl, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylsufinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, carbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amido, substituted or unsubstituted ureido, in which the alkyl, alkenyl and alkynyl portions contain from 1 up to about 14 carbon atoms, preferably from 1 to 6 atoms, and are either straight or branched chains or cyclic, and the aryl portions contain from about 4 to about 16 carbons, preferably 4 to 10 carbons; or

(ii) R¹² and R¹³ together (see, Formula IVb) are --CH₂ --, --CH═CH₂ -- O, S, NR¹⁴ in which R¹⁴ is H or alkyl, particularly lower alkyl. It is understood that in either (i) or (ii) each ring of A² in formula III may be unsubstituted or substituted with more than one substituent, each of which is selected independently from the selections set forth in (i) for R¹² and R¹³.

Among preferred embodiments herein, Ar² is has formula IV: ##STR5## in which X is O, NH or S; and R¹² and R¹³ are selected from H, lower alkyl, haloalkyl and halide. Again, it is understood that Ar² may be substituted with more than one substituent, each of which is selected independently from the selections set forth for R¹² and R¹³.

Of the compounds described herein, those that inhibit or increase an endothelin-mediated activity by about 50% at concentrations of less than about 100 μM are preferred. More preferred are those that inhibit or increase an endothelin-mediated activity by about 50% at concentrations of less than about 10 μM, more preferably less than about 1 μM and most preferably less than about 0.1 μM.

Among the most preferred compounds for use in the methods provided herein, are compounds that interact with ET_(B) receptors with an IC₅₀ of less than about 100 μM, preferably less than 10 μM, more preferably less than 1 μM, but with ET_(A) receptors with an IC₅₀ of greater than about 10 μM.

Among others of the preferred compounds for use in the methods herein are any compounds that interact with ET_(A) and/or ET_(B) receptors with an IC₅₀ of less than about 100 μM, more preferably less than 10 μM, and even more preferably less than about 1 μM, and most preferably less than about 0.1 μM.

Pharmaceutical compositions formulated for administration by an appropriate route and means containing effective concentrations of one or more of the compounds provided herein or pharmaceutically acceptable salts or acids thereof that deliver amounts effective for the treatment of hypertension, stroke, asthma, shock, ocular hypertension, glaucoma, renal failure, inadequate retinal perfusion and other conditions that are in some manner mediated by an endothelin peptide or that involve vasoconstriction or whose symptoms can be ameliorated by administration of an endothelin antagonist or agonist, are also provided. Particularly preferred compositions are those that deliver amounts effective for the treatment of hypertension or renal failure. The effective amounts and concentrations are effective for ameliorating any of the symptoms of any of the disorders.

Methods for treatment of endothelin-mediated disorders, including but not limited to, hypertension, asthma, shock, ocular hypertension, glaucoma, inadequate retinal perfusion and other conditions that are in some manner mediated by an endothelin peptide, or for treatment of disorder that involve vasoconstriction or that are ameliorated by administration of an endothelin antagonist or agonist are provided.

In particular, methods of treating endothelin-mediated disorders by administering effective amounts of the sulfonamides, prodrugs or other suitable derivatives of the sulfonamides are provided. In particular, methods for treating endothelin-mediated disorders, including hypertension, cardiovascular diseases, cardiac diseases including myocardial infarction, pulmonary hypertension, erythropoietin-mediated hypertension, respiratory diseases and inflammatory diseases, including asthma, bronchoconstriction, ophthalmologic diseases, gastroenteric diseases, renal failure, endotoxin shock, menstrual disorders, obstetric conditions, wounds, anaphylactic shock, hemorrhagic shock, and other diseases in which endothelin mediated physiological responses are implicated, by administering effective amounts of one or more of the compounds provided herein in pharmaceutically acceptable carriers are provided. Preferred methods of treatment are methods for treatment of asthma and other bronchial disorders in which ET_(B) receptors are implicated.

More preferred methods of treatment are those in which the compositions contain at least one compound that inhibits the interaction of an endothelin peptide with ET_(B) receptors at an IC₅₀ of less than about 10 μM, and preferably less than about 5 μM, more preferably less than about 1 μM, even more preferably less than 0.1 μM, and most preferably less than 0.05 μM More preferred methods are those in which the compositions contain at least one compound that inhibits the interaction of endothelin-1 with ET_(B) receptors at an IC₅₀ of less than about 100 and preferably less than about 50 μM, more preferably, less than about 10 μM, and even more preferably less than 1 μM, and most preferably less than about 0.1 μM, but do not inhibit binding of endothelinol to ET_(A) receptors at concentrations of about 10 μM or less.

More preferred methods are those in which the compositions contain at least one compound that inhibits the interaction of endothelin-1 with ET_(B) receptors at an IC₅₀ of less than about 100 μM, and preferably less than about 50 μM, more preferably less than about 10 μM, and most preferably less than about 1 μM, but that do not inhibit binding of endothelin-1 to ET_(A) receptors at concentrations of about 10 μM or less.

In practicing the methods, effective amounts of compositions containing therapeutically effective concentrations of the compounds formulated for oral, intravenous, local and topical application for the treatment of hypertension, cardiovascular diseases, cardiac diseases, including myocardial infarction, respiratory diseases, including asthma, inflammatory diseases, ophthalmologic diseases, gastroenteric diseases, renal failure, immunosuppressant-mediated renal vasoconstriction, erythropoietin-mediated vasoconstriction, endotoxin shock, anaphylactic shock, hemorrhagic shock, pulmonary hypertension, and other diseases in which endothelin mediated physiological responses are implicated are administered to an individual exhibiting the symptoms of one or more of these disorders. The amounts are effective to ameliorate or eliminate one or more symptoms of the disorders.

Methods for the identification and isolation of endothelin receptor subtypes are also provided. In particular, methods for detecting, distinguishing and isolating endothelin receptors using the disclosed compounds are provided. In particular, methods are provided for detecting, distinguishing and isolating endothelin receptors using the compounds provided herein.

In addition, methods for identifying compounds that are suitable for use in treating particular diseases based on their preferential affinity for a particular endothelin receptor subtype are also provided.

Articles of manufacture containing packaging material, a compound provided herein, which is effective for ameliorating the symptoms of an endothelin-mediated disorder, antagonizing the effects of endothelin or inhibiting binding of an endothelin peptide to an ET receptor with an IC₅₀ of less than about 10 μM, within the packaging material, and a label that indicates that the compound or salt thereof is used for antagonizing the effects of endothelin, treating an endothelin-mediated disorder, or inhibiting the binding of an endothelin peptide to an ET receptor are provided.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference. Such patents and publications represent the level of skill of those of skill in this art.

As used herein, endothelin (ET) peptides include peptides that have substantially the amino acid sequence of endothelin-1, endothelin-2 or endothelin-3 and that act as potent endogenous vasoconstrictor peptides.

As used herein, an endothelin-mediated condition is a condition that is caused by abnormal endothelin activity or one in which compounds that inhibit endothelin activity have therapeutic use. Such diseases include, but are not limited to hypertension, cardiovascular disease, asthma, inflammatory diseases, ophthalmologic disease, menstrual disorders, obstetric conditions, gastroenteric disease, renal failure, pulmonary hypertension, endotoxin shock, anaphylactic shock, or hemorrhagic shock. Endothelin-mediated conditions also include conditions that result from therapy with agents, such as erythropoietin and immunosuppressants, that elevate endothelin levels.

As used herein, an effective amount of a compound for treating a particular disease is an amount that is sufficient to ameliorate, or in some manner reduce the symptoms associated with the disease. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective. The amount may cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease. Typically, repeated administration is required to achieve the desired amelioration of symptoms.

As used herein, an endothelin agonist is a compound that potentates or exhibits a biological activity associated with or possessed by an endothelin peptide.

As used herein, an endothelin antagonist is a compound, such as a drug or an antibody, that inhibits endothelin-stimulated vasoconstriction and contraction and other endothelin-mediated physiological responses. The antagonist may act by interfering with the interaction of the endothelin with an endothelin-specific receptor or by interfering with the physiological response to or bioactivity of an endothelin isopeptide, such as vasoconstriction. Thus, as used herein, an endothelin antagonist interferes with endothelin-stimulated vasoconstriction or other response or interferes with the interaction of an endothelin with an endothelin-specific receptor, such as ET_(A) receptors, as assessed by assays known to those of skill in the art.

The effectiveness of potential agonists and antagonists can be assessed using methods known to those of skill in the art. For example, endothelin agonist activity can be identified by its ability to stimulate vasoconstriction of isolated rat thoracic aorta (Borges et 81. (1989) "Tissue selectivity of endothelin" Eur. J. Pharmacol. 165: 223-230). Endothelin antagonist activity can be assess by the ability to interfere with endothelin-induced vasoconstriction.

As used herein, the biological activity or bioactivity of endothelin includes any activity induced, potentiated or influenced by endothelin in vivo. It also includes both the ability to bind to particular receptors and to induce a functional response, such as vasoconstriction. These activities include, but are not limited to, vasoconstriction, vasorelaxation and bronchodilation. For example, ET_(B) receptors appear to be expressed in vascular endothelial cells and may mediate vasodilation and other such responses; whereas ET_(A) receptors, which are endothelin-1-specific, occur on smooth muscle and are linked to vasoconstriction Any assay known to those of skill in the art to measure or detect such activity may be used to assess such activity (see, e.g., Spokes et al. (1989) J. Cardiovasc. Pharmacol. 13(Suppl. 5):S191-S192; Spinella et al. (1991) Proc. Natl. Acad. Sci. USA 88: 7443-7446; Cardell et al. (1991) Neurochem. Int. 18:571-574); and the Examples herein).

As used herein, the IC₅₀ refers to an amount, concentration or dosage of a particular test compound that achieves a 50% inhibition of a maximal response, such as binding of endothelin to tissue receptors, in an assay that measures such response.

As used herein, EC₅₀ refers to a dosage, concentration or amount of a particular test compound that elicits a dose-dependent response at 50% of maximal expression of a particular response that is induced, provoked or potentiated by the particular test compound.

As used herein, pharmaceutically acceptable salts, esters or other derivatives of the compounds include any salts, esters or derivatives that may be readily prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects and that either are pharmaceutically active or are prodrugs. For example, hydroxy groups can be esterified or etherified.

As used herein, treatment means any manner in which the symptoms of a conditions, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein, such as use as contraceptive agents.

As used herein, amelioration of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.

As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.

As used herein, biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures.

As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound. To produce a prodrug, the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes. The prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design prodrugs of the compound (see, e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392). For example, succinyl-sulfathiazole is a prodrug of 4-amino-N-(2-thiazoyl)benzenesulfonamide (sulfathiazole) that exhibits altered transport characteristics.

As used herein, pseudohalides are compounds that behave substantially similar to halides. Such compounds can be treated as analogous with halides (X⁻, in which X is a halogen, such as Cl or Br). Pseudohalides include, but are not limited to cyanide, cyanate, thiocyanate selenocyanate and azide.

As used herein, lower alkyl, lower alkenyl, and lower alkynyl refer to carbon chains having less than about 6 carbons. In preferred embodiments of the compounds provided herein that include alkyl, alkenyl, or alkynyl portions include lower alkyl, lower alkenyl, and lower alkynyl portions.

As used herein aryl refers to cyclic groups containing from 3 to 15 carbon atoms, preferably from 5 to 10. The groups may be aromatic or saturated and includes groups, such as phenyl, substituted phenyl, naphthyl, substituted naphthyl, in which the substituent is lower alkyl, halogen, or lower alkoxy. Lower aryl refers to groups with about 3-6 carbons.

As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726).

A. Compounds for use in treating endothelin-mediated diseases

Compounds and methods for treating endothelin-mediated diseases using the compounds of formula I are provided. The compound of formula I provided herein are compounds of formula II in which Ar¹ is an isoxazolyl group: ##STR6## in which R¹ and R² are either (i), (ii) or (iii) as follows: (i) R¹ and R² are each independently selected from H, NH₂, NO₂, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkyloxy, haloalkyl, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylsufinyl, arylsulfonyl, aminocarbonyl, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amido, substituted or unsubstituted ureido, in which the alkyl, alkenyl and alkynyl portions are either straight or branched chains that contain from 1 up to about 10 carbon atoms and are be substituted or unsubstituted with substituents, such as those set forth above for R⁸ -R¹¹, and the aryl portions contain from about 4 to about 14 carbons, which also are unsubstituted or substituted; or,

(ii) R¹ and R² together form --(CH₂)_(n), where n is 1 to 4; or,

(iii) R¹ and R² together form --CH═CH--; and Ar² has the formula: ##STR7## in which: (i) R¹² and R¹³ are independently selected from H, NH₂, NO₂, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkyloxy, haloalkyl, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylsufinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amido, substituted or unsubstituted ureido, in which the alkyl, alkenyl and alkynyl portions contain from 1 up to about 14 carbon atoms, preferably from 1 to 6 atoms, and are either straight or branched chains or cyclic and are be unsubstituted or substituted with substituents, such as those set forth above for R⁸ -R¹¹, and the aryl portions contain from about 4 to about 16 carbons, preferably 4 to 10 carbons, and are unsubstituted or substituted with any of the above-noted groups; and X is O, NH, or S; or

(ii) R¹² and R¹³ together are O, S, or NR¹⁴ in which R¹⁴ is H or alkyl. It is understood that each ring of Ar² in formula V may be substituted with more than one substituent, each of which is selected independently from the selections set forth in (i) for R¹² and R¹³.

In certain preferred embodiments of the above compounds, R¹ and R² are selected independently from alkyl, lower alkenyl, lower alkynyl, lower haloalkyl, halide, pseudohalide and H; and R¹² and R¹³ are selected from H, lower alkyl, haloalkyl and halide.

In more preferred embodiments, R¹ and R² are independently selected from H, CH₃, C₂ H₅, H₂ C═CH, CH.tbd.C, Ph--O, Ph--CH₂, 4--CH₃ --C₆ H₄ O, halide, CF₃, C₂ F₅, n-C₃ H₇ and iso-C₃ H₇ ; and R¹² and R¹³ are independently selected from H, halide, NH₂, CF₃ CH₃, CN, CH₃, (CH₃)₃ C, C₅ H₁₁, CH₃ O, n-C₄ H₉ O and CH₂ ═CH.

In yet more preferred embodiments, R¹ is Br, Cl, H, CH₃, C₂ H₅, CF₃ ; R² is H, CH₃, C₂ H₅, or CF₃ ; R¹² and R¹³ are independently selected from H, CH₃, C₂ H₅, CF₃, and halide; and X is O.

Included among preferred compounds herein are the following:

N-(3,4-dimethyl-5-isoxazolyl)-4-biphenylsufonamide;

N-(4-bromo-3-methyl-5-isoxazolyl )-4-biphenylsulfonamide;

N-(4-methyl-3-trifluoromethyl-5-isoxazolyl)-4-biphenylsulfonamide;

N-(4-tridecyl-3-trifluoromethyl-5-isoxazolyl)-4-biphenylsulfonamide;

N-(4-chloro-3-methyl-5-isoxazolyl)-4-biphenylsulfonamide;

N-(4-bromo-5-methyl-3-isoxazolyl)-4-biphenylsulfonamide; and

N-(3,4-dimethyl-5-isoxazolyl )-2-dibenzofuransulfonamide.

Other preferred compounds are prodrugs of the compounds of formulas I and II and the especially preferred compounds or other derivatives, such as salts and acid derivatives, of the compounds of formulas I and II.

In assays that identify endothelin agonists and antagonists, compounds of formulas I and II inhibit binding of ET-1 to ET_(A) and/or ET_(B) receptors by 50% at concentrations less than about 100 μM, generally less than 10 μM and as low as concentrations less than 1 μM. Preferred compounds inhibit such binding at concentrations of less than about 10 μM. Most preferred compounds inhibit the binding at concentrations less than about 1 μM. Such compounds include the more preferred compounds herein and may be empirically identified by synthesizing and testing compounds as described herein.

The compounds provided herein are generally ET_(B) selective; i.e. the compounds provided herein inhibit binding of endothelin to ET_(B) receptors at concentrations about 10- about 100-fold less than they inhibit binding of endothelin to ET_(A) receptors.

B. Preparation of the compounds

The compounds, such as N-(3,4-dimethyl-5-isoxazolyl)biphenylsulfonamide (EXAMPLE 1), can be prepared from 4-biphenylsulfonyl chloride and an amino-substituted isoxazole, such as 5-amino-3,4-dimethylisoxazole, in dry pyridine (2.0 ml). Following the reaction, the pyridine is removed under reduced pressure and the residue is partitioned between water and ethyl acetate. The organic layer is washed and then dried over anhydrous magnesium sulfate, the solvents are evaporated and the residue is purified by column chromatography over silica gel (e.g., 1% methanol in chloroform as eluent) to yielded a solid. Further purification is achieved by recrystallization from ethyl acetate/hexanes, to yield the pure product. Alternatively, the sulfonamides can be prepared from the corresponding aminoisoxazole in tetrahydrofuran solution containing sodium hydride (see, e.g., EXAMPLE 7).

In some cases, the bis-sulfonyl compound is obtained as the major or exclusive product. The bis-sulfonated products can be readily hydrolyzed to the sulfonamide using aqueous sodium hydroxide and a suitable co-solvent, such as methanol or tetrahydrofuran, generally at room temperature

Prodrugs and other derivatives of the compounds suitable for administration to humans may also be designed and prepared by methods known to those of skill in the art (see, e,g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392.

The above-listed preferred compounds have been synthesized. Nuclear magnetic resonance spectroscopic (NMR), mass spectrometric, infrared spectroscopic and high performance liquid chromatographic analyses indicated that the synthesized compounds have structures consistent with those expected for such compounds and are at least about 98% pure.

C. Evaluation of the bioactivity of the compounds

Standard physiological, pharmacological and biochemical procedures are available for testing the compounds to identify those that possess any biological activities of an endothelin peptide or the ability to interfere with or inhibit endothelin peptides. Compounds that exhibit in vitro activities, such as the ability to bind to endothelin receptors or to compete with one or more of the endothelin peptides for binding to endothelin receptors can be used in the methods for isolation of endothelin receptors and the methods for distinguishing the specificities of endothelin receptors, and are candidates for use in the methods of treating endothelin-mediated disorders.

Thus, other preferred compounds of formulas I and II, in addition to those of specifically identified herein, that are endothelin antagonists or agonists may be identified using such screening assays.

1. Identifying compounds that modulate the activity of an endothelin peptide

The compounds are tested for the ability to modulate the activity of endothelin-1. Numerous assays are known to those of skill in the art for evaluating the ability of compounds to modulate the activity of endothelin (see, e.g., U.S. Pat. No. 5,114,918 to Ishikawa et al.; EP A1 0 436 189 to BANYU PHARMACEUTICAL CO., LTD. (Oct. 7, 1991); Borges et al. (1989) Eur. J. Pharm. 165: 223-230; Filep et al. (1991) Biochem. Biophys. Res. Commun. 177: 171-176). In vitro studies may be corroborated with in vivo studies (see, e.g., U.S. Pat. No. 5,114,918 to Ishikawa et al.; EP A1 0 436 189 to BANYU PHARMACEUTICAL CO., LTD. (Oct. 7, 1991)) and pharmaceutical activity thereby evaluated. Such assays are described in the Examples herein and include the ability to compete for binding to ET_(A) and ET_(B) receptors present on membranes isolated from cell lines that have been genetically engineered to express either ET_(A) or ET_(B) receptors on their cell surfaces.

The properties of a potential antagonist may be assessed as a function of its ability to inhibit an endothelin induced activity in vitro using a particular tissue, such as rat portal vein and aorta as well as rat uterus, trachea and vas deferens (see e,g., Borges, R., Von Grafenstein, H. and Knight, D. E., Tissue selectivity of endothelin, Eur. J. Pharmacol 165:223-230, (1989)). The ability to act as an endothelin antagonist in vivo can be tested in hypertensive rats, ddy mice or other recognized animal models (see, Kaltenbronn et al. (1990) J. Med. Chem. 33:838-845, see, also, U.S. Pat. No. 5,114,918 to Ishikawa et al.; and EP A1 0 436 189 to BANYU PHARMACEUTICAL CO., LTD (Oct. 7, 1991); see, also Bolger et al. (1983) J. Pharmacol. Exp. Ther. 225291-309). Using the results of such animal studies, pharmaceutical effectiveness may be evaluated and pharmaceutically effective dosages determined. A potential agonist may also be evaluated using in vitro and in vivo assays known to those of skill in the art,

Endothelin activity can be identified by the ability of a test compound to stimulate constriction of isolated rat thoracic aorta or portal vein ring segments (Borges et al. (1989) "Tissue selectivity of endothelin" Eur. J. Pharmacol. 165: 223-230). To perform the assay, the endothelium is abraded and ring segments mounted under tension in a tissue bath and treated with endothelin in the presence of the test compound. Changes in endothelin induced tension are recorded. Dose response curves may be generated and used to provide information regarding the relative inhibitory potency of the test compound. Other tissues, including heart, skeletal muscle, kidney, uterus, trachea and vas deferens, may be used for evaluating the effects of a particular test compound on tissue contraction.

Endothelin isotype specific antagonists may be identified by the ability of a test compound to interfere with endothelin binding to different tissues or cells expressing different endothelin-receptor subtypes, or to interfere with the biological effects of endothelin or an endothelin isotype (Takayanagi et al. (1991) Reg. Pep. 32: 23-37, Panek et al. (1992) Biochem. Biophys. Res. Commun. 183: 566-571). For example, ET_(B) receptors are expressed in vascular endothelial cells, possibly mediating the release of prostacyclin and endothelium-derived relaxing factor (De Nucci et al. (1988) Proc. Natl. Acad. Sci. USA 85:9797). ET_(A) receptors are not detected in cultured endothelial cells, which express ET_(A) receptors.

The binding of compounds or inhibition of binding of endothelin to ET_(B) receptors can be assessed by measuring the inhibition of endothelin-1-mediated release of prostacyclin, as measured by its major stable metabolite, 6-keto PGF_(1a), from cultured bovine aortic endothelial cells (see, e.g., Filep et al (1991) Biochem. and Biophys Res. Commun. 177: 171-176). Thus, the relative affinity of the compounds for different endothelin receptors may be evaluated by determining the inhibitory dose response curves using tissues that differ in receptor subtype.

Using such assays, the relative affinities of the compounds for ET_(A) receptors and ET_(B) receptors have been and can be assessed. Those that possess the desired properties, such as specific inhibition of binding of endothelin-1, are selected. The selected compounds that exhibit desirable activities may be therapeutically useful and are tested for such uses using the above-described assays from which in vivo effectiveness may be evaluated (see, e.g., U.S. Pat. Nos. 5,248,807; 5,240,910; 5,198,548; 5,187,195; 5,082,838; 5,230,999; published Canadian Application Nos. 2,067,288 and 2071193; published Great Britain Application No. 2,259,450; Published Internation PCT Application No. WO 93/08799; Benigi et al. (1993) Kidney International 44:440-444; and Nirei et al. (1993) Life Sciences 52:1869-1874). Compounds that exhibit in vitro activities that correlate with in vivo effectiveness will then be formulated in suitable pharmaceutical compositions and used as therapeutics.

The compounds also may be used in methods for identifying and isolating endothelin-specific receptors and aiding in the design of compounds that are more potent endothelin antagonists or agonists or that are more specific for a particular endothelin receptor.

2. Isolation of endothelin receptors

A method for identifying endothelin receptors is provided. In practicing this method, one or more of the compounds is linked to a support and used in methods of affinity purification of receptors. By selecting compounds with particular specificities, distinct subclasses of ET receptors may be identified,

One or more of the compounds may be linked to an appropriate resin, such as Affi-gel, covalently or by other linkage, by methods known to those of skill in the art for linking endothelin to such resins (see, Schvartz et al. (1990) Endocrinology 126: 3218-3222). The linked compounds can be those that are specific for ET_(A) or ET_(B) receptors or other subclass of receptors.

The resin is pre-equilibrated with a suitable buffer generally at a physiological pH (7 to 8). A composition containing solubilized receptors from a selected tissue are mixed with the resin to which the compound is linked and the receptors are selectively eluted. The receptors can be identified by testing them for binding to an endothelin isopeptide or analog or by other methods by which proteins are identified and characterized. Preparation of the receptors, the resin and the elution method may be performed by modification of standard protocols known to those of skill in the art (see, e.g., Schvartz et al. (1990) Endocrinology 126: 3218-3222).

Other methods for distinguishing receptor type based on differential affinity to any of the compounds herein are provided. Any of the assays described herein for measuring the affinity of selected compounds for endothelin receptors may also be used to distinguish receptors subtypes based on affinity for particular compounds provided herein. In particular, an unknown receptor may be identified as an ET_(A) or ET_(B) receptor by measuring the binding affinity of the unknown receptor for a compound provided herein that has a known affinity for one receptor over the other. Such preferential interaction is useful for determining the particular disease that may be treated with a compound prepared as described herein. For example, compounds with high affinity for ET_(A) receptors and little or no affinity for ET_(B) receptors are candidates for use as hypertensive agents; whereas, compounds that preferentially interact with ET_(B) receptors are candidates for use as anti-asthma agents.

D. Formulation and administration of the compositions

Effective concentrations of one or more of the sulfonamide compounds of formula I or II or pharmaceutically acceptable salts, esters or other derivatives thereof are mixed with a suitable pharmaceutical carrier or vehicle.

In instances in which the compounds exhibit insufficient solubility, methods for solubilizing compounds may be used. Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such as tween, or dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as salts of the compounds or prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.

The concentrations or the compounds are effective for delivery of an amount, upon administration, that ameliorates the symptoms of the endothelin-mediated disease. Typically, the compositions are formulated for single dosage administration.

Upon mixing or addition of the sulfonamide compound(s), the resulting mixture may be a solution, suspension, emulsion or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined.

Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.

The active compounds can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration. Preferred modes of administration include oral and parenteral modes of administration.

The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo systems (see, e.g., U.S. Pat. No. 5,114,918 to Ishikawa et al.; EP A1 0 436 189 to BANYU PHARMACEUTICAL CO., LTD (Oct. 7, 1991); Borges et al. (1989) Eur. J. Pharm. 165: 223-230;: Filep et al. (1991) Biochem. Biophys. Res. Commun. 177: 171-176) and then extrapolated therefrom for dosages for humans.

The concentration of active compound in the drug composition will depend on absorption, inactivation and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. For example, the amount that is delivered is sufficient to treat the symptoms of hypertension. The effective amounts for treating endothelin-mediated disorders are expected to be higher than the amount of the sulfonamide compound that would be administered for treating bacterial infections.

Typically a therapeutically effective dosage should produce a serum concentration of active ingredient of from about 0.1 ng/ml to about 50-100 μg/ml. The pharmaceutical compositions typically should provide a dosage of from about 0.01 mg to about 2000 mg of compound per kilogram of body weight per day. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

If oral administration is desired, the compound should be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.

Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules, For the purpose of oral therapeutic administration, the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules or troches. Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition.

The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder, such as microcrystalline cellulose, gum tragacanth and gelatin; an excipient such as starch and lactose, a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a gildant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, and fruit flavoring.

When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

The active materials can also be mixed with other active materials which do not impair the desired action, or with materials that supplement the desired action, such as antacids, H2 blockers, and diuretics. For example, if the compound is used for treating asthma or hypertension, it may be used with other bronchodilators and antihypertensive agents, respectively.

Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. Parental preparations can be enclosed in ampules, disposable syringes or multiple dose vials made of glass, plastic or other suitable material.

If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof. Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. For example, liposome formulations may be prepared as described in U.S. Pat. No. 4,522,811.

The active compounds may be prepared with carriers that protect the compound against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others. Methods for preparation of such formulations are known to those skilled in the art.

The compounds may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Such solutions, particularly those intended for ophthalmic use, may be formulated as 0.01%-10% isotonic solutions, pH about 5-7, with appropriate salts. The compounds may be formulated as aeorsols for topical application, such as by inhalation (see, e,g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment inflammatory diseases, particularly asthma).

Finally, the compounds may be packaged as articles of manufacture containing packaging material, a compound provided herein, which is effective for antagonizing the effects of endothelin or which inhibits binding of an endothelin peptide to an ET receptor with an IC₅₀ of less than about 10 μM, within the packaging material, and a label that indicates that the compound or salt thereof is used for antagonizing the effects of endothelin, treating endothelin-mediated disorders, or inhibiting the binding of an endothelin peptide to an ET receptor.

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLE 1

N-(3,4-Dimethyl-5-isoxazolyl)biphenylsulfonamide

(a) 4-Biphenylsulfonyl chloride

4-Biphenylsulfonic acid (3.0 g, 12.8 mmol) was heated at 70° C. with phosphorus oxychloride (1.30 ml, 14.0 mol) for 2 h. Excess phosphorus oxychloride was removed under reduced pressure. The residue was decomposed with ice water and extracted with ethyl acetate. The extract was washed with 5% sodium bicarbonate solution, dried over anhydrous magnesium sulfate and concentrated to yield 2.9 crude 4-biphenylsulfonyl chloride.

(b) N-(3,4-Dimethyl-5-isoxazolyl)biphenylsulfonamide

The 4-biphenylsulfonyl chloride from step (a) was added to a solution of 5-amino-3,4-dimethylisoxazole (250 mg, 2.2 mmol) and 4-(dimethyl)aminopyridine (5 mg) in dry pyridine (2.0 ml). The reaction mixture was stirred at room temperature for 4 h. Pyridine was removed under reduced pressure and the residue was partitioned between water and ethyl acetate. The organic layer was washed with 1N HCl (2×25 ml), brine (25 ml) and dried over anhydrous magnesium sulfate. Evaporation of the solvents left an oily residue that, after purification by column chromatography over silica gel (1% methanol in chloroform as eluent), yielded 337 mg (45%) white solid. Recrystallization from ethyl acetate/hexanes gave white crystals, m.p. 154°-155° C.

EXAMPLE 2

N-(4-Bromo-3-methyl-5-isoxazolyl)-4-biphenylsulfonamide

(a) 5-Amino-4-bromo-3-methylisoxazole

5-Amino-3-methylisoxazole (0.98 g, 10 mmol) was dissolved in chloroform (15 ml) and cooled to 0° C. N-Bromosuccinimide (1.78 g, 10 mmoles) was added in small portions over a period of 10 min. The stirring was continued for another 10 minutes at 0° C. The reaction mixture was diluted with chloroform (50 ml), washed with water (2×50 ml) and the organic layer was dried over magnesium sulfate. Removal of the solvent under reduced pressure gave the crude product, which was purified by column chromatography using 9:1, hexanes/ethyl acetate as the eluent, to give 5-amino-4-bromo-3-methylisoxazole (1.55 g, 87% yield ).

(b) N-(4-Biphenylsulfonyl)-N-(4-bromo-3-methyl-5-isoxazolyl)-4-biphenylsulfonamide

5-Amino-4-bromo-3-methylisoxazole (0.179 g, 1.0 mmol) was dissolved in dry pyridine (2 ml). 4-Biphenylsulfonyl chloride (0.509 g, 2.2 mmol) was added with stirring at ambient temperature. N,N-Dimethylaminopyridine (5 mg) was added, and stirring was continued at 50° C. for 16 h. The reaction mixture was diluted with dichloromethane (75 ml), washed with 1N HCl (2×50 ml) and the organic phase was dried over magnesium sulfate. The solvent was removed under reduced pressure to yield a crude product, which was purified by column chromatography using 8:2, hexanes/ethyl acetate, to give 0.390 g (60% yield) of N-(4-biphenylsulfonyl)-N-(4-bromo-3-methyl-5-isoxazolyl)-4-biphenylsulfonamide.

(c) N-(4-Bromo-3-methyl-5-isoxazolyl)-4-biphenylsulfonamide

N-(4-biphenylsuifonyl )-N-(4-bromo- 3-methyl- 5-isoxazolyl)-4-biphenylsulfonamide (0.150 g, 0.233 mmol) was dissolved in tetrahydrofuran (THF). Sodium hydroxide (0.120 g, 3.0 mmol) was added and the solution was warmed to 45° C. to dissolve the sodium hydroxide. Stirring was continued for 20 min. Tetrahydrofuran was removed under reduced pressure. The residue was dissolved in water, cooled to 0° C. and acidified to pH 3-4 with concentrated HCl. The solid precipitate was filtered off and dried in vacuo to give N-(4-bromo-3-methyl-5-isoxazolyl)-4-biphenylsulfonamide (94% yield), which was further purified by recrystallization from chloroform/hexanes, m.p. 133°-135° C.

EXAMPLE 3

N-(4-Methyl-3-trifluoromethyl-5-isoxazolyl)-4-biphenylsulfonamide

N-(4-Methyl- 3-trifluoromethyl-5-isoxazolyl)-4-biphenylsulfonamide was prepared in the same manner as described in Example 2b from 5-amino-4-methyl-3-trifluoromethyl-isoxazole and 4-biphenylsulfonyl chloride in 78% yield. Purification was achieved by recrystallization from methanol/water to give a white solid, m.p. 139°-140° C.

EXAMPLE 4

N-(4-Tridecyl-3-trifluoromethyl-5-isoxazolyl)-4-biphenylsulfonamide

N-(4-Tridecyl-3-trifluoromethyl-5-isoxazolyl)-4-biphenylsulfonamide was prepared, in the same manner as described in Example 2b, from 5-amino-4-tridecyl-3-trifluoromethyl-isoxazole and 4-biphenylsulfonyl chloride in 81% yield. Purification was achieved by recrystallization from methanol/water to give an off white solid, m.p. 115°-116° C.

EXAMPLE 5

N-(3,4-Dimethyl-5-isoxazolyl)-2-dibenzofuransulfonamide (IPI-528)

N-(3,4-Dimethyl-E-isoxazolyl)-2-dibenzofuransulfonamide was prepared, using the method described in Example 1b, from 5-amino-3,4-dimethylisoxazole and 2-benzofuransulfonyl chloride in 32% yield. Purification was achieved by recrystallization from chloroform/hexanes to give a white "cotton-like" solid, m.p. 173°-175° C. (dec.).

EXAMPLE 6

N-(4-Bromo-5-methyl-3-isoxazolyl)-4-sulfonamide

(a) 3-Amino-4-bromo-5-methylisoxazole

3-Amino-5-methylisoxazole (1.96 g, 20 mmol) was dissolved in chloroform (10 ml) and cooled to 0° C. N-Bromosuccinimide (3.56 g, 20 mmol) was added in small portions over a period of 10 min. The stirring was continued for another 15 minutes at 0° C. The reaction mixture was diluted with chloroform (100 ml), washed with water (2×50 ml) and the organic layer was dried over magnesium sulfate. Removal of the solvent under reduced pressure gave the crude product, which was purified by column chromatography, using 9:1 hexanes/ethyl acetate as the eluent, to give 3-amino-4-bromo-5-methylisoxazole (1.40 g, 40% yield).

(b) N-(4-bromo-5-methyl-3-isoxazolyl)-4-biphenylsulfonamide

N-(4-bromo-5-methyl-3-isoxazolyl)-4-biphenylsulfonamide was prepared, using the method in Example 1 b, from 3-amino-4-bromo-5-methylisoxazole and 4-biphenylsulfonyl chloride in 5% yield. The product (m.p. 154°-156° C.) was isolated in 51% yield by column chromatography, after recrystallization from ethyl acetate/hexanes. N-(4-Biphenylsulfonyl)-No(4-bromo-5-methyl-3-isoxazolyl)-4-biphenylsulfonamide was obtained in 51% yield.

EXAMPLE 7

N-(4-Chloro-3-methyl-5-isoxazolyl)-4-biphenylsulfonamide

(a) 5-Amino-4-chloro-3-methylisoxazole

Using the method in Example 2a, 5-amino-4-chloro-3-methylisoxazole was prepared from 5-amino-3-methylisoxazole and N-chlorosuccinimide in 90% yield.

(b) N-(4-Chloro-3-methyl-5-isoxazolyl)-4-biphenylsulfonamide

Sodium hydride (188 mg, 4.4 mmol) was suspended in dry THF (1 ml) and cooled to 0° C. A solution of 5-amino-4-chloro-3-methylisoxazole (mg, mmol) in dry THF (1 ml) was added with stirring. Once the addition was complete, the reaction mixture was warmed to room temperature for 10 min. The solution was recooled to 0° C., and 4-biphenylsulfonyl chloride (0.283 ml, 2.2 mmol) was added. Stirring was continued at 25° C. for 2 h. Excess sodium hydride was decomposed by the addition of methanol (0.4 ml) followed by water (0.5 ml). THF was removed under reduced pressure and the residue was dissolved in water (20 ml) and basified by addition of sodium hydroxide (pH 9-10). Neutral impurities were removed by extraction with ethyl acetate (2×10 ml). The aqueous layer was acidified to pH 2-3 using concentrated HCl and extracted with ethyl acetate (3×10 ml). The combined organic layer was dried over magnesium sulfate. Removal of the solvent gave N-(4-chloro-3-methyl-5-isoxazolyl)-4-biphenylsulfonamide in 83% yield. This product was purified by recrystallization from ethyl acetate/hexanes as a white solid, m.p. 129°-132° C.

EXAMPLE 8

Assays for identifying compounds that exhibit endothelin antagonistic and/or agonist activity

Compounds that are potential endothelin antagonists are identified by testing their ability to compete with ¹²⁵ I-labeled ET-1 for binding to human ET_(A) receptors or ET_(B) receptors present on isolated cell membranes. The effectiveness of the test compound as an antagonist or agonist of the biological tissue response of endothelin can also be assessed by measuring the effect on endothelin induced contraction of isolated rat thoracic aortic rings. The ability of the compounds to act as antagonists or agonists for ET_(B) receptors can be assess by testing the ability of the compounds are to inhibit endothelin-1 induced prostacyclin release from cultured bovine aortic endothelial cells.

A. Endothelin binding inhibition--Binding Test #1: Inhibition of binding to ET_(A) receptors

TE 671 cells (ATCC Accession No. HTB 139) express ET_(A) receptors. These cells were grown to confluence in T-175 flasks. Cells from multiple flasks were collected by scraping, pooled and centrifuged for 10 min at 190×g. The cells were resuspended in phosphate buffered saline (PBS) containing 10 mM EDTA using a Tenbroeck homogenizer. The suspension was centrifuged at 4° C. at 57,800×g for 15 min, the pellet was resuspended in 5 ml of buffer A (5mM HEPES buffer, pH 7.4 containing aprotinin (100 KIU/ml)) and then frozen and thawed once. 5 ml of Buffer B (5 mM HEPES Buffer, pH 7.4 containing 10 mM MnCl₂ and 0.001% deoxyribonuclease Type 1) was added, the suspension mixed by inversion and then incubated at 37° C. for 30 minutes. The mixture was centrifuged at 57,800×g as described above, the pellet washed twice with buffer A and then resuspended in buffer C (30 mM HEPES buffer, pH 7.4 containing aprotinin (100 KIU/ml) to give a final protein concentration of 2 mg/ml and stored at -70° C. until use.

The membrane suspension was diluted with binding buffer (30 mM HEPES buffer, pH 7.4 containing 150 mM NaCl, 5 mM MgCl₂, 0.5% Bacitracin) to a concentration of 8 μg/50 μl. ¹²⁵ I-endothelin-1 (3,000 cpm, 50 mL) was added to 50 μL of either: (A) endothelin-1 (for non specific binding) to give a final concentration 80 nM); (B) binding buffer (for total binding); or (C) a test compound (final concentration 1 nM to 100 μM). The membrane suspension (50 μL), containing up to 8 μg of membrane protein, was added to each of (A), (B), or (C). Mixtures were shaken, and incubated at 4° C. for 16-18 hours, and then centrifuged at 4° C. for 25 min at 2,500×g. The supernatant, containing unbound radioactivity, was decanted and the pellet counted on a Genesys multiwell gamma counter. The degree of inhibition of binding (D) was calculated according to the following equation: ##EQU1## Each test was generally performed in triplicate. B. Endothelin binding inhibition--Binding Test #2: Inhibition of binding to ET_(B) receptors

COS7 cells were transfected with DNA encoding the ET_(B) receptor, The resulting cells, which express the human ET_(B) receptor, were grown to confluence in T-150 flasks. Membrane was prepared as described above. The binding assay was performed as described above using the membrane preparation diluted with binding buffer to a concentration of 1 μg/50 μl.

The COS7 cells, described above, that had been transfected with DNA encoding the ET_(B) receptor and express the human ET_(B) receptor on their surfaces were grown to confluence in T-175 flasks. Cells from multiple flasks were collected by scraping, pooled and centrifuged for 10 min. at 190×g. The cells were resuspended in phosphate buffered saline (PBS) containing 10 mM EDTA using a Tenbroeck homogenizer. The suspension was centrifuged at 4° Ct 57,800×g for 15 min, the pellet was resuspended in 5 ml of buffer A (5 mM HEPES buffer, pH 7.4 containing aprotinin (100 KIU/ml)) and then frozen and thawed once. Five ml of Buffer B (5 mM HEPES Buffer, pH 7.4 containing 10 mM MnCl₂ and 0.001% deoxyribonuclease Type 1) was added, the suspension mixed by inversion and then incubated at 37° C. for 30 minutes. The mixture was centrifuged at 57,800×g as described above, the pellet washed twice with buffer A and then resuspended in buffer C (30 mM HEPES buffer, pH 7.4 containing aprotinin (100 KIU/ml) to give a final protein concentration of 2 mg/ml.

The binding assay was performed as described above using the membrane preparation diluted to give 1 μg/50 μl of binding buffer.

C. Test for activity against endothelin-induced contraction of isolated rat thoracic aortic rings

The effectiveness of the test compound as an antagonist or agonist of the biological tissue response of endothelin can assessed by measuring the effect on endothelin induced contraction of isolated rat thoracic aortic rings (see, e.g., Borges et al. (1989) Eur. J. Pharmacol. 165:223-230) or by measuring the ability to contract the tissue when added alone.

Compounds to be tested are prepared as 100 μM stocks. If necessary to effect dissolution, the compounds are first dissolved in a minimum amount of DMSO and diluted with 150 mM NaCl. Because DMSO can cause relaxation of the aortic ring, control solutions containing varying concentrations of DMSQ were tested.

The thoracic portion of the adult rat aorta is excised, the endothelium abraded by gentle rubbing and then cut into 3 mm ring segments. Segments are suspended under a 2 g preload in a 10 ml organ bath filled with Krebs'-Henseleit solution saturated with a gas mixture of 95% O₂ and 5% CO₂ (118 mM NaCl, 4,7 mM KCl; 1.2 mM MgSO₄, 1.2 mM KH₂ PO₄, 25 mM NaHCO₃, 2.5 mM CaCl₂, 10 mM D-glucose) gassed with 95% O_(2/5) % CO₂. Changes in tension are measured isometrically and recorded using a Grass Polygraph coupled to a force transducer.

Endothelin is added to the organ bath in a cumulatively increasing manner, and the effects of the test compounds on the concentration-response curve for endothelin-1 are examined. Compounds are added 15 min prior to the addition of endothelin-1.

D. Assay for identifying compounds that have agonist and/or antagonistic activity against ET_(B) receptors

1. Stimulation of prostacyclin release

Since endothelin-1 stimulates the release of prostacyclin from cultured bovine aortic endothelial cells, the compounds that have agonist or antagonist activity are identified by their ability to inhibit endothelin-1 induced prostacyclin release from such endothelial cells by measuring 6-keto PGF_(1a) substantially as described by (Filep et al. (1991) Biochem. Biophys. Res. Commun. 177 171-176. Bovine aortic cells are obtained from collagenase-treated bovine aorta, seeded into culture plates, grown in Medium 199 supplemented with heat inactivated 15% fetal calf serum, and L-glutamine (2 mM), penicillin, streptomycin and fungizone, and subcultured at least four times. The cells are then seeded in six-well plates in the same medium. Eight hours before the assay, after the cells reach confluence, the medium is replaced. The cells are then incubated with a) medium alone, b) medium containing endothelin-1 (10 nM), c) test compound alone, and d) test compound+endothelin-1 (10 nM).

After a 15 min incubation, the medium is removed from each well and the concentrations of 6-keto PGF_(1a) are measured by a direct immunoassay. Prostacyclin production is calculated as the difference between the amount of 6-keto PGF_(1a) released by the cells challenged with the endothelin-1 minus the amount released by identically treated unchallenged cells. Compounds that stimulate 6-keto PGF_(1a) release possess agonist activity and those which inhibit endothelin-1 6-keto PGF_(1a) release possess antagonist activity.

2. Inhibition of sarafotoxin 6c induced contraction

Sarafotoxin 6c is a specific ET_(B) antagonist that contracts rat fundal stomach strips. The effectiveness of tests compounds to inhibit this sarafotoxin 6c-induced contraction of rat fundal stomach strips is used as a measure ET_(B) antagonist activity. Two isolated rat fundal stomach strips are suspended under a 1 g load in a 10 ml organ bath filled with Krebs'-Henseleit solution containing 10 μM cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123; see, U.S. Pat. No. 5,114,918 to Ishikawa et al.), 5 μM indomethacin, and saturated with a gas mixture of 95% O₂ /5% CO₂. Changes in tension are measured isometrically and recorded using a Grass Polygraph coupled to a force transducer. Sarafotoxin 6c is added cumulatively to one strip while the second strip is preincubated for 15 min with a test compound prior to addition of cumulative doses of sarafotoxin 6c. The effects of the test compounds on the concentration-response curve for sarafotoxin 6c are examined.

E. Results

The IC₅₀ for each of the compounds of Examples 1-7 for ET_(A) and/or ET_(B) receptors has been measured. All of the compounds have an IC₅₀ of less than 100 μM for either or both of the ET_(A) and ET_(B) receptors. Many of the compounds have an IC₅₀ less than about 10 μM and others have an IC₅₀ less than about 1 μM. The compounds appear to be selective for ET_(B) receptors in that they have an IC₅₀ for ET_(B) receptors that is substantially less (10 to 100-fold or more) than for ET_(A) receptors.

Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims. 

We claim:
 1. A compound of formula I: ##STR8## or a pharmaceutically acceptable salt of a compound of formula I, wherein: R¹ and R² are either (i), (ii) or (iii) as follows:(i) R¹ and R² are each independently selected from the group consisting of H, NH₂, NO₂, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkyloxy, haloalkyl, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylsufinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amido and substituted or unsubstituted ureido, in which the alkyl, alkenyl and alkynyl portions contain from 1 up to about 14 carbon atoms and are either straight or branched chains, and the aryl portions contain from about 4 to about 16 carbons; or, (ii) R¹ and R² together form --(CH₂)_(n), where n is 1 to 4; or, (iii) R¹ and R² together form --CH═CH--; Ar² is a group of formula: ##STR9## ps in which (i) R¹² and R¹³ are independently selected from the group consisting of H, OH NH S, NH₂, NO₂, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, dialkylamino, alkylthio, alkyloxy, haloalkyl, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylsufinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, carbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amido, substituted or unsubstituted ureido, in which the alkyl, alkenyl and alkynyl portions contain from 1 up to about 14 carbon atoms, preferably from 1 to 6 atoms, and are either straight or branched chains or cyclic, and the aryl portions contain from about 4 to about 16 carbons, preferably 4 to 10 carbons; or(ii) R¹² and R¹³ together are O, S, or NR¹⁴ in which R¹⁴ is H or alkyl; and each 6-membered ring of Ar² in formula II is unsubstituted or substituted with one to six substituents that are each independently selected from R¹² or R¹³, as defined in (i).
 2. The compounds of claim 1, wherein R¹ and R² are selected independently from the group consisting of lower alkyl, lower alkenyl, lower alkynyl, lower haloalkyl, halide, pseudohalide and H; and R¹² and R¹³ are selected from H, lower alkyl, haloalkyl and halide.
 3. The compounds of claim 1, wherein R¹ and R² are independently selected from the group consisting of H, CH₃, C₂ H₅, H₂ C═CH, CH.tbd.C, Ph--O, Ph--CH₂, 4--CH₃ --C₆ H₄ O, halide, CF₃, C₂ F₅, n--C₃ H₇ and iso-C₃ H₇ ; and R¹² and R¹³ are each independently selected from the group consisting of H, halide, NH₂, alkylamino, dialkylamino, CF₃ CH₃, CN, CH₃, (CH₃)₃ C, C₅ H₁₁, CH₃ O, n--C₄ H₉ O, CO₂ H, and CH₂ ═CH.
 4. The compounds of claim 1, wherein R¹ is Br, Cl, H, CH₃, C₂ H₅ or CF₃ ; R² is H, CH₃, C₂ H₅, or CF₃ ; R¹² and R¹³ are independently selected from H, CH₃, C₂ H₅, CF₃, or halide; and X is O.
 5. The compounds of claim 1, wherein Ar² has formula II: ##STR10## in which X is O, NH or S; and R¹² and R¹³ are selected from the group consisting of H, lower alkyl, haloalkyl and halide.
 6. The compounds of claim 5, wherein R¹ and R² are independently selected from the group consisting of H, CH₃, C₂ H₅, H₂ C═CH, CH.tbd.C, Ph--O, Ph--CH₂, 4--CH₃ --C₆ H₄ O, halide, CF₃, C₂ F₅, n--C₃ H₇ and iso-C₃ H₇ ; and R¹² and R¹³ are each independently selected from the group consisting of H, halide, NH₂, CF₃ CH₃, CN, CH₃, (CH₃)₃ C, C₅ H₁₁, CH₃ O, n--C₄ H₉ O and CH₂ ═CH.
 7. The compounds of claim 5, wherein R¹ is Br, Cl, H, CH₃, C₂ H₅ or CF₃ ; R² is H, CH₃, C₂ H₅, or CF₃ ; R¹² and R¹³ are independently selected from H, CH₃, C₂ H₅, CF₃, or halide; and X is O.
 8. The compounds of claim 2, wherein Ar² has formula II: ##STR11## in which X is O, NH or S.
 9. The compounds of claim 8, wherein R¹ and R² are independently selected from the group consisting of H, CH₃, C₂ H₅, H₂ C═CH, CH.tbd.C, Ph--O, Ph--CH₂, 4--CH₃ --C₆ H₄ O, halide, CF₃, C₂ F₅, n--C₃ H₇ and iso-C₃ H₇ ; and R¹² and R¹³ are each independently selected from the group consisting of H, halide, NH₂, CF₃ CH₃, CN, CH₃, (CH₃)₃ C, C₅ H₁₁, CH₃ O, n--C₄ H₉ O and CH₂ ═CH.
 10. The compounds of claim 8, wherein R¹ is Br, Cl, H, CH₃, C₂ H₅ or CF₃ ; R² is H, CH₃, C₂ H₅, or CF₃ ; R¹² and R¹³ are independently selected from H, CH₃, C₂ H₅, CF₃, or halide; and X is O.
 11. The compounds of claim 1 that are selected from the group consisting of N-(3,4-dimethyl-5-isoxazolyl)-4-biphenylsufonamide, N-(4-bromo-3-methyl-5-isoxazolyl)-4-biphenylsulfonamide, N-(4-methyl-3-trifluoromethyl-5-isoxazolyl)-4-biphenylsulfonamide, N-(4-tridecyl-3-trifluoromethyl-5-isoxazolyl)-4-biphenylsulfonamide, N-(4-chloro-3-methyl-5-isoxazolyl)-4-biphenylsulfonamide, N-(4-bromo-5-methyl-3-isoxazolyl)-4-biphenylsulfonamide and N-(3,4-dimethyl-5-isoxazolyl)-2-dibenzofuransulfonamide.
 12. The compound of claim 1 that is N-(3,4-dimethyl-5-isoxazolyl)-4-biphenylsufonamide.
 13. The compound of claim 1 that is N-(4-bromo-3-methyl-5-isoxazolyl)-4-biphenylsulfonamide.
 14. The compound of claim 1 that is N-(4-methyl-3-trifluoromethyl-5-isoxazolyl)-4-biphenylsulfonamide.
 15. The compound of claim 1 that is N-(4-tridecyl-3-trifluoromethyl-5-isoxazolyl)-4-biphenylsulfonamide.
 16. The compound of claim 1 that is N-(4-chloro-3-methyl-5-isoxazolyl)-4-biphenylsulfonamide.
 17. The compound of claim 1 that is N-(4-bromo-5-methyl-3-isoxazolyl)-4-biphenylsulfonamide.
 18. The compound of claim 1 that is N-(3,4-dimethyl-5-isoxazolyl)-2-dibenzofuransulfonamide.
 19. A pharmaceutical composition, comprising a compound of claim 1 in a pharmaceutically acceptable carrier.
 20. A pharmaceutical composition, comprising a compound of claim 11 in a pharmaceutically acceptable carrier.
 21. A pharmaceutical composition formulated for single dosage administration, comprising an effective amount of one or more compounds of claim 1 or pharmaceutically acceptable salts of the compounds of claim 1, wherein the amount is effective for ameliorating the symptoms of an endothelin-mediated disease.
 22. An article of manufacture, comprising packaging material and a compound of claim 1 or pharmaceutically acceptable salt of a compound of claim 1 contained within the packaging material, wherein the compound or salt therof is effective for antagonizing the effects of endothelin, ameliorating the symptoms of an endothelin-mediated disorder, or inhibiting the binding of an endothelin peptide to an ET receptor with an IC₅₀ of less than about 10 μM; and the packaging material includes a label that indicates that the compound or salt thereof is used for antagonizing the effects of endothelin, inhibiting the binding of endothelin to an endothelin receptor or treating an endothelin-mediated disorder.
 23. The compounds of claim 1 that are 4-biphenylsulfonamides.
 24. The compounds of claim 23, wherein R¹ and R² are independently selected from the group consisting of H, CH₃, C₂ H₅, H₂ C═CH, CH.tbd.C, Ph--O, Ph--CH₂, 4--CH₃ --CH₆ H₄ O, halide, CF₃, C₂ F₅, n--C₃ H₇ and iso-C₃ H₇ ; and R¹² and R¹³ are each independently selected from the group consisting of H halide, NH₂, CF₃ CH₃, CH, CH₃, (CH₃)₃ C, C₅ H₁₁, CH₃ O, n--C₄ H₉ O and CH₂ ═CH.
 25. The compounds of claim 23, wherein R¹ is Br, Cl, H, CH₃, C₂ H₅ or CF₃ ; R² is H, CH₃, C₂ H₅, or CF₃ ; R¹² and R¹³ are independently selected from H, CH₃, C₂ H₅, CF₃, or halide; and X is O. 